Recently, an immunoassay of a strip type for immunochromatography has increased its versatility as a simple in vitro diagnostic kit or portable diagnostic device for detecting a substance to be detected (an antigen or the like) in a sample liquid by utilizing the specific reactivity of an antibody.
Incidentally, in an immunochromatography detection method for detecting a substance to be detected (an antigen or the like) by sandwiching the substance by an antibody, in the case where the substance to be detected (the antigen or the like) is present in a test sample in a large excess amount, a problem that a false-negative phenomenon (also referred to as “prozone phenomenon” or “high-dose hook effect”) occurs as if apparently, the substance to be detected is present in a small amount or is not present at all, and not only is the S/N ratio at the time of detection decreased, but also an erroneous test result is caused has been well known, and studies have been made for excluding this prozone phenomenon.
In order to solve this problem, in an immunochromatography method utilizing an affinity chromatography principle, a sandwich method in which a substance to be detected (an antigen or the like) is sandwiched by an antibody to the substance and an antibody bound to a fine particle and detected, a method in which a substance to be detected (an antigen or the like) or an equivalent is immobilized on a chromatography support and made to compete with the substance to be detected (the antigen or the like), whereby a prozone phenomenon characteristic of the sandwich method is suppressed and the objectivity of determination is ensured, and moreover, a technique for excluding a prozone phenomenon by including a free antibody have been developed.
For example, in an immunological quantitative device, by setting a sample addition part, a labeled reactant-coated part downstream thereof, a part having a target substance (a substance to be detected) or a substance equivalent thereto immobilized thereon further downstream thereof, and a part having antibodies immobilized thereon in the form of a plurality of ladders further downstream thereof, a competitive reaction is allowed to proceed on a chromatography support, and also by providing free antibodies in the form of a plurality of ladders, a prozone phenomenon is suppressed/excluded. Then, a quantitative device which enables rapid measurement objectively in one step by making a determination based on the number of reaction lines or the coloring pattern thereof by the immobilized antibodies has been proposed (see Patent Document 1).
Further, in an affinity chromatography detection method, a method in which on a developing support, a first detection region having a substance which specifically binds to a detection target substance immobilized thereon and a second detection region having a substance which competes with the detection target substance immobilized thereon are provided, a developing solution containing a substance which is labeled with a labeling substance and specifically binds to the detection target substance and a sample is developed through these detection regions, and the detection target substance is detected based on a signal in the first and second detection regions, whereby a false-negative phenomenon is less likely to occur, and the occurrence of prozone is avoided has been proposed (see Patent Document 2).
Then, another method for excluding a prozone phenomenon, the provision of a test strip having an expanded dynamic range and a detection method using the strip have been proposed, and according to this, a prozone sample can be detected without diluting the sample.
For example, in Patent Document 3, a test strip which is a chromatography strip provided with a first end and a second end including at least a first reaction region and a second reaction region containing a capture agent which specifically binds to an analyte, and including an absorption pad in the first end, wherein the absorption pad enables the lateral flow of a sample, whereby the capture agent can bind to at least part of the analyte, and a method for detecting and measuring the presence of an analyte based on a signal of the first reaction region or from the strength of a signal from the first reaction region, the second reaction region, or a combination thereof have been proposed (see Patent Document 3).
However, the method of the related art which is an immunochromatography method in which an antibody is labeled with an insoluble support (colloidal gold particles, colored latex particles, or the like) (also referred to as “particle immunochromatography method”), and excludes a prozone phenomenon has an advantage that a quantitative determination can be achieved based on the number of reaction lines or the coloring pattern thereof for a high concentration analyte sample in which a detection target substance (an antigen or the like) is present in a large excess amount by charging free antibodies in the form of a plurality of ladders, or by providing a second or more detection regions, but has a problem that the coloration of a low concentration analyte is reduced or the free antibodies are denatured, and therefore, an effect of the measures for a prozone phenomenon is reduced over time.
Further, the method of the related art for excluding a prozone phenomenon is mainly a method capable of objectively determining/measuring a detection target substance by charging free antibodies in the form of a plurality of ladders, or by providing a second or more detection regions in addition to a standard first detection region for a detection target substance which is present in a large excess amount, and making a comprehensive determination based on these signals.
However, since there are a plurality of detection lines or there are at least two detection regions, the determination criteria are not simple but are inevitably complicated, and therefore, the method has a problem that not everyone can perform the method easily without error, and therefore, a method capable of simply making a determination has been demanded.